We have described key features of the core transcriptional regulatory circuitry of human and murine embryonic stem cells (ESCs) and identified fundamental regulatory mechanisms that contribute to the control of gene expression programs in mammalian cells. Several research groups have recently discovered that master transcription factors bind large clusters of transcriptional enhancers, called super- enhancers, which drive expression of genes that play key roles in the control of transcriptional regulatory circuitry and cell identity. A substantial portion of disease-associated variation occurs in these super- enhancers. Thus, super-enhancers play important roles in the control of cellular identity in both normal and disease states. However, gaps in our understanding of super-enhancer structure and function limit our understanding their roles in control of cell state, in development and in disease. We propose to expand our understanding of the transcriptional control of mammalian cell identity by further investigating super- enhancer components and functions in ESCs and other clinically important cell types. In addition, super- enhancers suggest a new approach to investigate the regulatory circuitry of clinically important cells, and we propose to test this approach. To accomplish these goals, the specific aims of the proposal are: 1) Further investigate the structure and function of super-enhancers, 2) Determine whether the noncoding RNA produced from super-enhancers contributes to enhancer function in ESCs, 3) Investigate the dynamics of super-enhancer decommissioning and establishment during differentiation, and 4) Investigate the regulatory circuitry of clinically important cell types through super-enhancer analysis. Improved understanding of super-enhancers should continue to produce new insights into the control of cell state, reveal the key themes that operate to control gene expression programs in mammalian cell types, and produce new insights into disease mechanisms. These studies should also provide the foundation for understanding transcriptional regulation in a broad spectrum of normal and diseased cells and for cellular reprogramming of clinically important cells.